Project Summary This is a competitive renew of a grant that has uncovered the essential roles of a heat shock protein (HSP) gp96 (also known as grp94 and HSP90b1) in chaperoning multiple TLRs in the endoplasmic reticulum. TLRs are critical sensors for pathogens in the initiation of innate immunity and for subsequent activation of adaptive immunity. Until recently, research in TLR biology has been focused primarily on downstream signaling events. It is increasingly appreciated that TLR biogenesis and trafficking are also tightly regulated to prevent inflammation and autoimmunity. My laboratory is at the forefront to address the function of gp96 using genetic approaches. We have discovered and published in the last funding period that: (i) gp96 is a master chaperone for both cell surface and intracellular TLRs; (ii) gp96 is not required for the assembly of immunoglobulin but it plays critical roles in the proper compartmentalization of B1 cells and marginal zone B cells due to its ability to also chaperone integrins; (iii) the Drosophila gp93 is the gp96 ortholog in that it can rescue the expression of both TLRs and integrins in gp96 KO murine cells; (iv) gp96 is critical for T and B lymphopoiesis but not myelopoiesis; (v) the folding of TLRs by gp96 is dependent on a substrate (TLR)-specific co-chaperone, CNPY3 (also known as PRAT4A). Unexpectedly, we have made two additional novel observations. First, gp96 plays critical roles in canonical Wnt signaling such that when gp96 is deleted from the gut, mice develop a profound and lethal enterocolitis. Second, when gp96 was selectively knocked down from macrophages, we observed a significant attenuation of inflammation-associated colon cancer and a concurrent reduction of IL23 and IL17. We thus hypothesize that gp96 provide a crucial link for multiple processes including inflammation, tissue homeostasis and cancer. We will tackle our hypothesis with the following three specific aims: (1) Determine the molecular mechanisms of gp96 in folding TLRs with specific focus on the roles of co-chaperones. (2) Establish the roles and mechanisms of gp96 in gut homeostasis. (3) Dissect the macrophage-intrinsic roles and mechanisms of gp96 in inflammation-associated colon cancer. Successful execution of our proposal shall significantly advance the field of chaperone biology, innate immunity, inflammation and tumor immunology.